This application reports the isolation and characterization of canine interleukin-5 (xe2x80x9cIL-5xe2x80x9d) and includes nucleic and amino acid sequences therefor. More particularly, it concerns a nucleic acid sequence which comprises DNA which encodes canine interleukin-5.
Interleukin 5
Interleukin-5 (IL-5) has been studied in both human and murine systems. It was initially designated T cell-replacing factor or B cell growth factor II (BCGF II). IL-5 is understood to induce or mediate multiple effects. It promotes the proliferation of activated B lymphocytes as well as the generation of both IgM and IgG responses. IL-5 also promotes IgA secretion, apparently by acting on cells that already express surface IgA.
IL-5 cDNA clones from mouse and human species have been isolated. See Takatsu, K., and Tominaga, A., xe2x80x9cInterleukin 5 and its Receptorxe2x80x9d, Progress in Growth Factor Research, 3: 87-102 (1991), incorporated herein by reference. Mouse IL-5 consists of 133 amino acid residues, including a signal sequence of 21 residues and three sites for N-glycosylation. In contrast, human IL-5 consists of 139 amino acid residues, including a signal sequence of 22 residues and two sites for N-glycosylation. Both mouse and human IL-5 exist as a dimer linked by disulfide bond. Further, mouse and human IL-5 are 71% homologous at the amino acid level. However, while human IL-5 is capable of stimulating mouse cells, mouse IL-5 is only weakly cross-reactive with human cells.
Prior to this invention, no canine IL-5 DNA or amino acid sequence has been reported. It is unknown whether canine IL-5 will cross react with human or mouse cells.
In Vitro Activities
In humans IL-5 is known to exert most of its biological activities on hematopoietic lineages outside the lymphoid compartment. This cytokine acts as an eosinophil stimulating factor; e.g., it augments the number of eosinophil colonies that develop in semisolid bone marrow cultures. IL-5 is a potent regulator of eosinophilia and appears to act on relatively mature progenitors, causing them to proliferate and to differentiate into mature effector cells. In fact, eosinophils induced in vitro by IL-5 are fully functional and have been demonstrated to kill antibody-coated schistosomula of Schistosoma mansoni and antibody-coated tumor cells.
Since IL-5 plays a major role in the regulation of eosinophils that are prominently involved in allergic inflammation, IL-5 inhibition may have potential therapeutic benefits for various allergies. See Cuss, xe2x80x9cInhibition of Interleukin-5 with a Monoclonal Antibody Attenuates Allergic Inflammation,xe2x80x9d Allergy, 52: 787-794 (1997), incorporated herein by reference.
IL-5 may also act in concert with other hemtopoietic cytokines such as IL-3 and GM-CSF. The three are known for increasing oxidative metabolism, membrane receptor expression and the release of granule proteins as well as for their role in inducing eosinophilopoiesis. In vitro data shows that IL-5 acts in synergy with other activation signals, as in the case of IL-5 and immune complexes. See Desremeux, P. and Capron, M., xe2x80x9cEosinophils in Allergic Reactionsxe2x80x9d, Current Opin. in Immunol., 8: 790-795 (1996), incorporated herein by reference.
The other known activities of IL-5 all relate to regulation of B-cell immune responses. IL-5 was first described as a T-cell activity that induced antigen-stimulated murine B cells to differentiate into both IgM- and IgG-secreting plasma cells. See Takatsu, K., and Tominaga, A., supra. Subsequent studies have shown that IL-5 is the major factor inducing differentiation to immunoglobulin (Ig) production in B cells which were activated by contact with activated T helper cells. Murine IL-5 also augments proliferation of and induces secretion of Ig in a number of B-cell lines. Further, it has similar activity on xe2x80x9cin vivo-activatedxe2x80x9d normal B cells. Murine IL-5 can also enhance the production of IgA in LPS-stimulated B-cell cultures; however, its principal activity is not as a switch-inducing factor, and it does not specifically enhance IgA in T-cell stimulated cultures. Murine IL-5 induces expression of the P55 chain of the IL-2 receptor on normal B cells and, in combination with IL-4, renders these cells responsive to IL-2 stimulation.
Presently, however, the role of IL-5 in human B-cell growth and differentiation remains controversial. IL-5 is inactive in many of the culture systems commonly used to assay human B-cell growth factors and differentiation factors. However, in other assay systems, IL-5 has been shown to be active on human B cells activated with mitogens or activated T-cell clones. Thus, the contribution of IL-5 to the helper activity of T cells in humans is presently not understood.
In vitro biological activities similar to those described above in humans can be postulated for IL-5 in dogs.
In Vivo Activities
In humans, IL-5 appears to be the most specific cytokine for activation of eosinophils. IL-5, IL-3, and GM-CSF may also act in concert to activate eosinophils and basophils, as these three cytokines cross-react on receptors because each cytokine shares a common chain. These cytokines have been identified in late phase reactions in humans. See Desremaux, P., and Capron, M, xe2x80x9cEosinophils in Allergic Reactionsxe2x80x9d, Current Opin. in Immunol., 8: 790-795 (1996).
IL-5 is involved in the eosinophilia that develops during parasitic infections and allergic reactions, but not in the IgG1 or IgE antibody responses associated with these infections. Administration of anti-IL-5 monoclonal antibody blocks the development of both blood and tissue eosinophil responses, but fails to affect IgG1 or IgE secretion. See Cuss, D., xe2x80x9cInhibition of Interleukin-5 with a Monoclonal Antibody Attenuates Allergic Inflammationxe2x80x9d, Allergy, 52: 787-794 (1997).
In vitro experiments suggested that IL-5 is an eosinophil regulator, and numerous in vivo experiments indicate that IL-5 is, in fact, a predominant regulator of eosinophilia. This was readily seen in a series of experiments where the administration of anti-IL-5 antibodies to mice infected with Nippostrongylus brasiliensis, Schistosoma mansoni, Heligmosomoides polygyrus, or Strongyloides venezuelensis totally blocked the development of eosinophilia. Consistent with these data, mice that have over expressed IL-5 due to the introduction of IL-5 transgenes or IL-5 retroviral constructs are characterized by dramatic increases in eosin counts. In contrast to IL-5 expressing mice, mice that have been genetically engineered to over express either IL-3 or GM-CSF live only a few weeks due to massive tissue infiltration and destruction by the greatly increased numbers of myeloid cells.
In vivo biological activities similar to those described above in humans can be postulated for IL-5 in dogs.
The present invention relates in part to recombinant DNA molecules, and conservative variants thereof, that encode canine IL-5. Disclosed is an isolated polynucleotide comprising the nucleotide sequence of canine IL-5 of FIG. 1 (SEQ ID NO:1) or its complement.
Also disclosed is an isolated canine IL-5 nucleic acid sequence comprising around 80%, preferably around 85%, preferably around 90%, more preferably around 95%, even more preferably around 97% and most preferably around 99% homology to a nucleic acid sequence of canine IL-5 of FIG. 1 (SEQ ID NO:1) or its complement.
Further disclosed is an isolate canine IL-5 nucleic acid comprising the nucleotide sequence encoding the polypeptide comprising the amino acid sequence of FIG. 1.
Also disclosed is an isolated canine IL-5 polynucleotide comprising around 80%, preferably around 85%, preferably around 90%, more preferably around 95%, even more preferably around 97% and most preferably around 99% homology to a nucleic acid comprising the nucleotide sequence encoding the polypeptide comprising the amino acid sequence of FIG. 1.
Further disclosed is a canine IL-5 polynucleotide comprising at least about 20 consecutive nucleotides, preferably at least about 30 consecutive nucleotides, more preferably at least about 50 consecutive nucleotides, more preferably at least about 100 consecutive nucleotides, more preferably at least about 150 consecutive nucleotides, more preferably at least about 200 consecutive nucleotides, more preferably at least about 250 consecutive nucleotides, more preferably at least about 300 consecutive nucleotides, more preferably at least about 350 consecutive nucleotides, most preferably at least about 400 consecutive nucleotides, of the sequence of FIG. 1, and conservative variants thereof.
These polynucleotides are referred to herein as canine IL-5 polynucleotides.
In another aspect, an isolated canine IL-5 polypeptide comprising the amino acid sequence of FIG. 1 is disclosed.
Also disclosed is an isolated canine IL-5 polypeptide comprising around 80%, preferably around 85%, preferably around 90%, more preferably around 95%, even more preferably around 97%, and most preferably around 99% identity to the canine IL-5 polypeptide of FIG. 1.
Also disclosed is a canine IL-5 polypeptide comprising at least about 7 consecutive amino acids, preferably at least about 10 consecutive amino acids, more preferably at least about 33 consecutive amino acids, more preferably at least about 50 consecutive amino acids, more preferably at least about 66 consecutive amino acids, more preferably at least about 83 consecutive amino acids, more preferably at least about 99 consecutive amino acids, more preferably at least about 116 consecutive amino acids, most preferably at least about 132 consecutive amino acids, of the sequence of FIG. 1 and conservative variants thereof.
In another aspect of the present invention, polypeptides produced using a recombinant expression vector containing a canine IL-5 sequence are set forth.
The above-identified polypeptides are herein referred to as canine IL-5 polypeptides.
In a related aspect, a method is described that uses a polynucleotide encoding canine IL-5 to produce an IL-5 polypeptide. The method comprises expressing the nucleic acid molecule in a transformed host cell and purifying the IL-5 protein, either from the cells or cell debris, or from the medium if the polypeptides are secreted.
In another aspect of the present invention, purified canine IL-5 is provided.
In another aspect of the present invention, recombinant vectors, such as expression vectors, comprising a DNA sequence encoding canine IL-5 are disclosed.
In a further aspect of the present invention, cells comprising the recombinant vectors, which themselves comprise a DNA sequence encoding canine IL-5, are set forth.
In a further related aspect, a method for producing canine IL-5 is disclosed that comprises the steps of inserting a transcription regulatory sequence proximal to the IL-5 gene in a cell comprising that gene, and stimulating production of IL-5 through the regulatory sequence.
In an additional aspect of the present invention, a canine IL-5 polypeptide can be linked to a multiply antigenic peptide so that the multiply antigenic peptide comprises multiple copies of the same peptide or of various peptides. The various peptides can comprise conservative variants of peptides of the invention.
Disclosed is a peptide in accordance with the invention, or a conservative variant thereof, that can be linked to a plant virus particle so that the particle comprises multiple copies of the peptide or of various peptides, in accordance with the invention. The various peptides can comprise conservative variants of peptides of the invention.
Also disclosed are antibodies and other specific binding molecules that bind to canine IL-5 and to mimetopes of IL-5 epitopes.
Disclosed is a method for generating canine auto-antibodies directed to the canine IL-5 molecule, said method comprising providing a peptide or a conservative variant thereof, in accordance with the invention, and administering the provided peptide to a dog. The method can further comprise a step of mixing the provided peptide with an adjuvant prior to the administering step, wherein the administering step comprises administering the mixture of the peptide and the adjuvant. The above method may be useful in the treatment and prophylaxis of eosinophil/IL-5 mediated allergic responses in dogs.
Amino acids: The basic building blocks of proteins, having an amino end and a carboxyl end for the formation of peptide bonds with neighboring amino acids.
Polypeptide: A linear series of amino acids connected one to the other by peptide bonds between the amino and carboxyl groups of adjacent amino acids. As used herein, the term xe2x80x9cpolypeptidexe2x80x9d includes peptides and proteins.
Conservative amino acid substitutions: Substitutions that do not substantially affect the character of the polypeptide. Suitable amino acid variations do not appreciably alter the function of the polypeptide of the invention, although the level of activity may be altered. Activity can be measured in an in vitro test for IL-5 activity, such as an assay for stimulation of eosinophils in culture. The effect of the variations can be reduced by selecting for polypeptide variants that have a minimum of changes in regions of high homology between the canine, mouse and human IL-5 molecules, and are thus conserved regions of the polypeptide. Further, substitutions that replace amino acid(s) with those with similar physical characteristics are preferred. Table 2 lists some preferred amino acid substitutions.
Conservative amino acid additions or deletions: Additions or deletions of amino acids that take place outside of the sequences conserved between canine, human and mouse IL-5 genes and that do not appreciably alter the function of the polypeptide of the invention, although the level of activity may be altered.
Conservative amino acid variants: Amino acid sequences that result from conservative amino acid substitutions.
Nucleotide: A monomeric unit of DNA or RNA nucleic acid sequence consisting of a sugar moiety (pentose), a phosphate, and a nitrogenous heterocyclic base. The four DNA bases are adenine (xe2x80x9cAxe2x80x9d), guanine (xe2x80x9cGxe2x80x9d), cytosine (xe2x80x9cCxe2x80x9d) and thymine (xe2x80x9cTxe2x80x9d). The four RNA bases are A, G, C and uracil (xe2x80x9cUxe2x80x9d). Nucleotides A and G are purines, whereas C, T, and U are pyrimidines.
Polynucleotide: A linear series of nucleotides connected one to the other by phosphodiester bonds between the 3xe2x80x2 and 5xe2x80x2 carbons of adjacent pentoses.
Conservative nucleotide substitutions: Nucleotide substitutions that either do not result in changes in the amino acid sequence or that result in conservative amino acid substitutions.
Conservative nucleotide additions or deletions: Additions or deletions of groups of 3 nucleotides (codons) that do not cause a frameshift in translation of a polynucleotide, and that do not create or delete stop codons. These additions or deletions take place outside of the nucleotide sequences conserved between canine, human and mouse IL-5 genes.
Conservative nucleic acid variants: Nucleotide sequences that result from conservative nucleotide substitutions or conservative nucleotide additions or deletions.
Recombinant DNA Molecule: A hybrid DNA sequence comprising at least two nucleotide sequences, the first sequence not normally being found together in nature with the second.
Vector: A plasmid, phage DNA or other DNA sequence, able to replicate in a host cell and capable of carrying an exogenously added DNA sequence for purposes of amplification or expression of the added DNA sequence.
Expression Control Sequence: A DNA sequence of nucleotides that controls and regulates expression of structural genes when operatively linked to those genes.
Regulatory Sequence: A DNA sequence that is necessary for regulation of expression of a coding sequence to which the DNA sequence is operably associated. The nature of regulatory sequences varies depending upon the host organism. For instance, in prokaryotes, regulatory sequences include a promoter, and/or a transcription termination sequence. In eucaryotes, such regulatory sequences include a promoter and/or a transcription termination sequence, and may also include a secretory leader sequence for secretion of a polypeptide attached to the leader.
Mimetope: A variant of the epitope of an antibody, selected by its ability to be bound by antibodies that cross-react with the first antibody.
Pharmaceutically acceptable carrier: Any carrier that is used by persons in the art for administration into a human that does not itself induce any undesirable side effects such as the production of antibodies, fever, etc. This term includes excipients.
Purified or isolated: The molecule so indicated is present in substantial absence of other biological macromolecules of the same species or type.
Specific binding: Binding of one substance to another at a greater binding affinity than background binding. Two substances which exhibit specific binding are referred to as specific binding partners, or as a specific binding pair. An antibody and its antigen are one example of a specific binding pair.
Specific Binding Molecule: A molecule which exhibits specific binding to its corresponding binding partner to form a specific binding pair. As used herein, this definition of specific binding molecule includes but is not limited to monoclonal and polyclonal antibodies, antigen-binding fragments of these antibodies, hybrid antibodies, single-chain antibodies, and recombinant molecules capable of specific binding to a ligand.
Therapeutically effective amount: The amount that is effective for production of a desired result. This amount varies depending upon the health and physical condition of the animal being treated, the capacity of the animal""s immune system to synthesize antibodies, the degree of protection desired, the formulation, and other pertinent factors.